Much of the progress in basic chemistry has been motivated by the interest in the nature of disease in the human. Disease impairs the performance of vital functions. Diabetes, heart disease, cancer, liver ailments, and infection are just a few disorders that interfere with normal bodily functions. Since the 1950's, there has been an unprecedented growth in the number of laboratory tests available to a physician to detect such disorders. Limitations such as sensitivity, specificity, and accuracy vary from one test to the next.
Enzymes, the catalysts which promote almost all biochemical reactions, exist in all body organs, and within the cells of these organs. Healthy cells are semipermeable, allowing small molecules to pass through the cell membrane, but retaining large molecules, such as enzymes. However, as organ cells are damaged, for example, through disease, the cell's permeability increases, allowing small quantities of enzymes to leak into the blood stream, where they may be found by chemical analysis.
The determination of enzyme activity in the subject animal or human patient's body fluids (generally blood serum) leads to an appreciation of the extent and nature of organ damage. This determination is made very sensitive and highly specific by using the native catalytic properties of the enzyme molecule to convert substrate to product. In general, if a particular enzyme is present in a sample in amounts exceeding a recognized norm, this may indicate one or more physical ailments. Thus, an abnormal level of certain enzymes acts as a fingerprint for certain diseases.
In order to measure the amount of enzyme present in a sample, it is necessary to choose a particular substrate and reaction conditions that favor the evaluation of the activity of the enzyme of interest. Reaction conditions are chosen to permit the enzyme of interest to catalyze the substrate to product reaction. The substrate and other necessary chemicals are mixed with a sample containing an unknown quantity of the enzyme of interest and the reaction is allowed to proceed. The amount of product formed in a given time is proportional to enzyme concentration. For a particular enzyme, the measurement of activity may be indicative of cell damage. The measurement of enzymes that exist in only one organ provide a clear chemical indicator of that organ's health. For example, the measurement of specific heart, liver, bone, prostate and pancreatic enzymes is possible.
The determination of enzymes is most frequently performed in clinical laboratories by experienced technicians using automated instruments incorporating light absorption, fluorescence or electrochemical detection capabilities.
Enzymes possess certain characteristics not common to other types of catalysts. First, they are quite sensitive to small changes in temperature. Second, they often show sharp changes in activity as the pH of the system changes. Third, enzymes may be very specific in catalyzing a particular type of reaction. Enzymatic specificity is necessary to maintain some degree of independence of all the reactions occurring in complex organisms. Last, many enzymes differ from other catalysts in that they are more efficient. The greatest similarity that enzymes and nonbiochemical catalysts have in common is that they change the rate of the overall chemical reaction.
The rate of a chemical reaction is expressed as a change in concentration of reactant or product in a given time interval. Enzymes usually enhance reaction rates by at least a millionfold. At a constant concentration of an enzyme, the reaction rate increases with increasing substrate (reactant) concentration until a maximal velocity is reached. In contrast, uncatalyzed reactions do not show this effect.
This property of being saturable is expressed mathematically in the Michaelis-Menten Model for enzyme kinetics. Basically, "saturable" means that the velocity of the reaction being catalyzed by the enzyme is first order, or linearly dependent on substrate concentration at lower substrate concentrations, but the velocity of the reaction becomes nearly zero order, or independent of substrate concentrations, at higher substrate concentrations, or when all enzyme active sites are saturated with substrate.
The Michaelis-Menten model provides a basis for understanding the kinetic properties of many enzymes. The general reaction scheme that this model follows is: ##STR1## An enzyme (E) combines with substrate (S) to form an enzyme substrate complex (ES). This complex can proceed to form a product (P) or dissociate to E and S. The equation (2) ##EQU1## describes the rate of product formation, V. V.sub.Max is the maximum rate which occurs when the enzyme is fully saturated with substrate. K.sub.M is the substrate concentration at which the reaction is one half the maximal rate. Closer examination of this model reveals that the maximal velocity is the product of k.sub.3 and the total enzyme concentration. k.sub.3 is the turnover number that can be further defined as the number of substrate molecules converted into product per unit time. Equation (2) shows that for a given enzyme, the rate of product formation will differ for various concentrations of that enzyme.
Rates of product formation can be determined by measurement of an instrument signal that is proportional to the concentration of product. The method commonly used for measuring the concentration of product is absorption spectrometry. Some enzymatic reactions are coupled with a "coenzyme" such as NAD which when reduced to NADH absorbs light at 340 nm. The changes in the absorbance of the solution due to the NADH are monitored. The amount of NADH produced can be directly related to the activity of an enzyme present in the assay.
At high substrate concentrations, K.sub.M is &lt;&lt;[S] and equation 2 becomes: EQU V=V.sub.Max ( 3)
This equation shows that at high substrate concentrations, the reaction velocity is zero order in S. This relationship is represented by the portion of the curve between c and d in FIG. 1. When zero order conditions prevail, the velocity of the reaction is solely determined by the concentration (activity) of the enzyme.
Zero order conditions are generally used in the measurement of enzyme concentration (when the enzyme concentration is variable) while first order conditions (section a-b in FIG. 1) are used in the measurement of reactant species (when the enzyme concentration is constant).
In order to measure V, the rate of product formation, it is necessary according to known methods to use an instrument, such as a spectrometer, which measures the change in absorptivity over time, which corresponds to the amount of enzyme in the sample.
Although the instrument measuring method performed in clinical laboratories is acceptable, there are situations in which access to a suiteable laboratory and/or use of an instrument for measuring enzyme concentrations is impossible or impractical, making a non instrumental and generally extralaboratory method highly desirable. For example, non instrumental, generally extralaboratory, methods may be useful in physicians' offices, in tests performed by non-skilled individuals on themselves at home, in the field for testing animals or humans away from modern facilities, at sea, under battle conditions and in a variety of situations when testing is desirable but clinical laboratory instrumental testing methods are unavailable.
Several examples of testing may include the measurement of acetylcholinesterase (E.C. 3.1.1.8) in the blood of farmers or pesticide applicators or in the blood of soldiers exposed to nerve gas toxins, the measurement of the alanine aminotransferase (ALT, E.C. 2.6.1.2) in the blood of potential blood donors as a surrogate test for hepatitis, the measurement of amylase (E.C. 3.2.1.1) in the blood serum of sailors having abdominal pain and suspected of having pancreatitis, or the measurement of creatine kinase (E.C. 2.7.3.2) in the blood serum of persons having chest pain and suspected of having a myocardial infarction.
A non-instrumental device could be used in Third World countries lacking instrumentation, where the device would be valuable in the initial diagnosis and the monitoring of disease. Such a device could also be used for a number of different medical applications other than those dealing with disease.